Fluorescence-based cell-based primary high throughput screening assay to identify antagonists of the orexin 1 receptor (OX1R; HCRTR1)
Name: Fluorescence-based cell-based primary high throughput screening assay to identify antagonists of the orexin 1 receptor (OX1R; HCRTR1). ..more
BioActive Compounds: 1387
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Patricia McDonald, TSRI
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R01 DA023915-02
Grant Proposal PI: Patricia McDonald
External Assay ID: OX1R_ANT_FLUO8_1536_1X%INH PRUN
Name: Fluorescence-based cell-based primary high throughput screening assay to identify antagonists of the orexin 1 receptor (OX1R; HCRTR1).
Heterotrimeric G-protein coupled receptors (GPCRs) are major targets for disease therapeutics, due in part to their broad tissue distribution, structural diversity, varied modes of action, and disease association (1-4). Most non-sensory GPCRs are expressed in the brain and regulate critical neuronal functions involved in feeding, sleep, mood, and addiction (5, 6). For example, in the later al hypothalamic region of the brain, two orexin neuropeptides (orexin A and orexin B) derived from proteolytic processing of the same orexin precursor (7), signal through the Gq-coupled GPCRs OX1R and OX2R to stimulate food consumption (8, 9). OX1R binds orexin A selectively, while OX2R binds both orexin A and orexin B. Recently, signaling by orexin A through OX1R has been shown to play a critical role in cocaine-seeking behavior (10) and morphine withdrawal (6). Additional studies reveal OX1R involvement in behavioral plasticity (11), the sleep-wake cycle (12, 13), and gastric acid secretion (14), and that OX1R may bind other neuropeptides such as neuropeptide Y and secretin (15). As a result, the identification of a selective OX1R antagonist would serve as a useful tool for exploring orexin biology, and the role of OX1R in drug addiction.
1. Bosier, B and Hermans, E, Versatility of GPCR recognition by drugs: from biological implications to therapeutic relevance. Trends Pharmacol Sci, 2007. 28(8): p. 438-46.
2. Lagerstrom, MC and Schioth, HB, Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov, 2008. 7(4): p. 339-57.
3. Pan, HL, Wu, ZZ, Zhou, HY, Chen, SR, Zhang, HM and Li, DP, Modulation of pain transmission by G-protein-coupled receptors. Pharmacol Ther, 2008. 117(1): p. 141-61.
4. Thompson, MD, Cole, DE and Jose, PA, Pharmacogenomics of G protein-coupled receptor signaling: insights from health and disease. Methods Mol Biol, 2008. 448: p. 77-107.
5. Gainetdinov, RR, Premont, RT, Bohn, LM, Lefkowitz, RJ and Caron, MG, Desensitization of G protein-coupled receptors and neuronal functions. Annu Rev Neurosci, 2004. 27: p. 107-44.
6. Sharf, R, Sarhan, M and Dileone, RJ, Orexin mediates the expression of precipitated morphine withdrawal and concurrent activation of the nucleus accumbens shell. Biol Psychiatry, 2008. 64(3): p. 175-83.
7. de Lecea, L, Kilduff, TS, Peyron, C, Gao, X, Foye, PE, Danielson, PE, Fukuhara, C, Battenberg, EL, Gautvik, VT, Bartlett, FS, 2nd, Frankel, WN, van den Pol, AN, Bloom, FE, Gautvik, KM and Sutcliffe, JG, The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci U S A, 1998. 95(1): p. 322-7.
8. Sakurai, T, Amemiya, A, Ishii, M, Matsuzaki, I, Chemelli, RM, Tanaka, H, Williams, SC, Richardson, JA, Kozlowski, GP, Wilson, S, Arch, JR, Buckingham, RE, Haynes, AC, Carr, SA, Annan, RS, McNulty, DE, Liu, WS, Terrett, JA, Elshourbagy, NA, Bergsma, DJ and Yanagisawa, M, Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell, 1998. 92(4): p. 573-85.
9. Hara, J, Beuckmann, CT, Nambu, T, Willie, JT, Chemelli, RM, Sinton, CM, Sugiyama, F, Yagami, K, Goto, K, Yanagisawa, M and Sakurai, T, Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity. Neuron, 2001. 30(2): p. 345-54.
10. Smith, RJ, See, RE and Aston-Jones, G, Orexin/hypocretin signaling at the orexin 1 receptor regulates cue-elicited cocaine-seeking. Eur J Neurosci, 2009. 30(3): p. 493-503.
11. Winrow, CJ, Tanis, KQ, Reiss, DR, Rigby, AM, Uslaner, JM, Uebele, VN, Doran, SM, Fox, SV, Garson, SL, Gotter, AL, Levine, DM, Roecker, AJ, Coleman, PJ, Koblan, KS and Renger, JJ, Orexin receptor antagonism prevents transcriptional and behavioral plasticity resulting from stimulant exposure. Neuropharmacology, 2009.
12. Dugovic, C, Shelton, JE, Aluisio, LE, Fraser, IC, Jiang, X, Sutton, SW, Bonaventure, P, Yun, S, Li, X, Lord, B, Dvorak, CA, Carruthers, NI and Lovenberg, TW, Blockade of orexin-1 receptors attenuates orexin-2 receptor antagonism-induced sleep promotion in the rat. J Pharmacol Exp Ther, 2009. 330(1): p. 142-51.
13. Hagan, JJ, Leslie, RA, Patel, S, Evans, ML, Wattam, TA, Holmes, S, Benham, CD, Taylor, SG, Routledge, C, Hemmati, P, Munton, RP, Ashmeade, TE, Shah, AS, Hatcher, JP, Hatcher, PD, Jones, DN, Smith, MI, Piper, DC, Hunter, AJ, Porter, RA and Upton, N, Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc Natl Acad Sci U S A, 1999. 96(19): p. 10911-6.
14. Eliassi, A, Nazari, M and Naghdi, N, Role of the ventromedial hypothalamic orexin-1 receptors in regulation of gastric Acid secretion in conscious rats. J Neuroendocrinol, 2009. 21(3): p. 177-82.
15. Kane, JK, Tanaka, H, Parker, SL, Yanagisawa, M and Li, MD, Sensitivity of orexin-A binding to phospholipase C inhibitors, neuropeptide Y, and secretin. Biochem Biophys Res Commun, 2000. 272(3): p. 959-65.
orexin, Orexin 1 receptor, OX1, OX1R, OXR1, hypocretin-1 receptor, hypocretin, HCRTR1, HCRTR-1, GPCR, antagonism, inhibition, inhibit, inhibitor, addiction, relapse, cocaine, substance abuse, brain, CHO cells, Fluo8, Fluo-8, fluorescence, fluorescent, dye, primary, primary screen, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that inhibit orexin 1 receptor (OX1R; HCRTR1) activity. In this assay CHO cells stably transfected with the human OX1R receptor (CHO/OX1R cell line) are treated with test compounds, followed by measurement of intracellular calcium as monitored by the FLUO-8 fluorescent, cell permeable calcium indicator dye. As designed, compounds that act as OX1R antagonists will decrease calcium mobilization, resulting in decreased relative fluorescence of the indicator dye, and thus decreased well fluorescence. Compounds are tested in singlicate at a nominal concentration of 5 uM.
The CHO/OX1R cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 25 mM HEPES, 739 ug/mL Geneticin, , and 1X antibiotic mix (penicillin, streptomycin, and neomycin).
The day before the assay 2000 cells in 3 uL of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 degrees C, 5% CO2, and 95% RH for 23 hours. Next, 2 uL of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer's protocol) was added to each well. After incubation for 1 hour at 37 degrees C, 5% CO2, and 95% RH, 25 nL of test compound in DMSO, or DMSO alone were dispensed to the appropriate wells. The assay was started after an additional 30 minute incubation at room temperature, by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 6 seconds on the FLIPR Tetra (Molecular Devices). Next, 15 nL of Orexin-A agonist in DMSO (EC77 average response), or DMSO alone were dispensed to the appropriate wells. Then a real time fluorescence measurement was immediately performed for the remaining 94 seconds of the assay.
A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:
Ratio = I_Max / I_Min
I_Max represents the maximum measured fluorescence emission intensity over the 100 second read and,
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.
Percent inhibition was calculated from the median ratio as follows:
% Inhibition = ( 1 - ( ( Ratio Test_Compound - Median_Ratio_High_Control ) / ( Median_Ratio_Low_Control - Median_Ratio_High_Control ) ) ) * 100
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing Orexin A peptide Challenge.
High_Control is defined as wells containing the antagonist SB-334867-A.
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.
PubChem Activity Outcome and Score:
The PubChem Activity Score range for active compounds is 100-28, for inactive 62-0.
List of Reagents:
CHO/OX1R cells (supplied by Assay Provider)
Fluo-8 No Wash Calcium Assay Kit (AAT Bioquest, part 36316)
Trypan red plus (ABD Bioquest, part 2456)
Ham's F-12 media (Invitrogen, part 11765-054)
Geneticin (Invitrogen, part 10131-027)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16140-071)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Nunc, part 159910)
Agonist: Orexin A peptide (Tocris, part 1455)
Antagonist: SB-334867-A (Tocris, part 1960)
1536-well plates (Greiner, part 789072)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)